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Gonadal Histo-Morphology and Antifertility Effects of Bonny

Light Crude Oil in Male Rats

Victor A. Fischer¹*, Christie E. Fischer¹, Mfon Akpaso¹, Oladipo A. Ashiru²

1. Department of Anatomy, Faculty of Basic Medical Sciences, University of Calabar, Calabar- Nigeria.

2. Assisted Reproductive Technology (ART) Centre, Maryland, Lagos – Nigeria.

*Email of corresponding author: victorfischer52000@yahoo.com

Abstract

The study investigated the antifertility effects of ingestion of Bonny Light Crude Oil (BLCO) at a dose of 0.5,

1.5 2.5 and 3.5ml/kg body weight in adult male rats following oral administration for 60days. The animals were

sacrificed, the epididymal spermatozoa expressed out and a homogenate made for semen analysis. The testes

were removed for histological processing. The sperm count was significantly reduced (21.80±0.57, 19.00±0.85,

16.20±0.00 and 12.90±0.99 million/ml respectively) when compared to their corresponding control groups

26.30±1.27, 25.50±0.71, 26.70±2.69, and 25.60±0.57 million/ml) at P˂0.05. Degenerative and necrotic changes

of cells in the seminiferous tubules and interstitium was observed in the histology of the testes. BLCO has

adverse effects on male reproductive system. This may imply possible antifertility for male rats exposed to

BLCO ingestion.

Keywords: Bonny light crude oil, testes, antifertility, male rat

1. Introduction

Crude oil is a mixture of hydrogen molecules, which are organic compounds of carbon and hydrogen atoms that

may include from one to sixty carbons (Petroleum Handbook, 1996). It contains trace amount of sulphur

containing chemicals, such as sulfides, mercaptans, thiophenes, and other more complex compounds. Although

the chemical composition of the crude oil varies by source, crude oil and petroleum products share certain toxic

characteristics and are toxic to biological life (Walkinson and Holt,1987)). The oil industry is the backbone of

the Nigerian economy, accounting for over 90% of the total foreign exchange revenue. Nigeria’s marker crude

oil on the international market is the Bonny Light Crude Oil (BLCO). It is preferred to others because of its low

specific gravity and low corrosiveness to refinery infrastructure. Industrialization, through exploitation and

exploration of crude oil or total petroleum hydrocarbon (TPH), has introduced into the ecosystem substances that

are potentially toxic to life and environment (Dede and Kagbo, 2002).

The magnitude of oil pollution and damage occasioned by multinational oil companies operating in the Niger

delta region of Nigeria is not only disturbing but incredible (Akpofure et al., 2000). Bioaccumulation of crude oil

in marine life possess potential health hazards to terrestrial species (Shore and Douben, 1994). There is available

evidence suggesting changes in chemical properties of soil following contamination by crude oil (Ifeadi and

Nwankwo, 1987). The devastating consequences of the spill of crude oil and its products in the Niger delta

region with its eventual hazards on both aerial and terrestrial environs tantamount to an irreversible chain effect

both on biodiversity and human safety (Akpofure et al., 2000).

Many studies on the toxic effects of crude oil of different geological formation in laboratory and non-laboratory

animals have been carried out (Payne et al., 1987; 1983; Rice et al., 1977). Epidemiological data and results of

toxicity studies in experimental animals consistently report that there is significant health risk due to prolonged

exposure to petroleum (Didia et al., 2003; Sheepers and Bios, 1992). The chemical composition of crude oil

differs widely and these chemicals are capable of mimicking the inherent actions of reproductive hormones and

hence have the ability to disrupt the neuro-endocrine system or the functions of the gonads directly (Colborn et

al., 1993).

Some people (Kalabari’s, Ijaw’s, Ogoni’s) ingest crude oil for various medicinal purposes; as laxative, anti-

poisoning agent, anti-convulsion agent, used for treatment of arthritis, snake antidote, especially in rural areas

where the conventional antidotes are not available (Dede et al., 2002). Animal studies have shown cyto toxic

effects on some organs from exposure to crude oil compounds (Didia et al., 2003, Eyong et al.,2004; Eyong,

2000; Khan et al., 1999) and gonads contain rapidly proliferating cells that are probably susceptible to damage

by PAH’s (ASTDR, 2004).

This research work was therefore undertaken to investigate the histological changes and antifertility effects

associated with ingestion of BLCO on male wistar rats.

2. Materials And Methods

2.1 Bonny Light Crude Oil

The bonny light crude oil used for this study was obtained from Shell Petroleum Development Company, Port

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Harcourt, Rivers state with authority from the Department of Petroleum Resources, NNPC, Lagos Nigeria.

2.2 Experimental Animals

Sixty (60) male albino wistar rats obtained from the animal house of the Department of pharmacology,

University of Calabar were used for this study. The animals whose weight ranged from 150 – 200g were

acclimatized for three (3) weeks before the commencement of the experiment in the animal house of the

Department of Human Anatomy. The animals were housed in well-ventilated cages and kept under controlled

environmental conditions of temperature (25±5°c), relative humidity (50±5%) and 12-hour light/dark cycle. The

animals were fed with grower’s marsh and tap water ad libitum

2.3 Experimental Design

The animals were assigned into four (4) parallel groups A, B, C, and D consisting of ten (10) experimental

animals and five (5) control animals, the doses used were based on predetermined LD50 values obtained from

previous studies (Eyong, 2000). The experimental animals in groups A, B, C, and D received 0.5, 1.5, 2.5 and

3.5ml/kg B.W of BLCO respectively, the control animals in the corresponding groups received 0.5, 1.5, 2.5 and

3.5ml/kg B.W of normal saline respectively both by oral gastric intubation. The treatment was done once a day

on alternate days for a period of sixty (60) days. At the end of the sixty (60) days, the animals were euthanized

under chloroform vapor and sacrificed. The spermatozoa was expressed out, a homogenate was made and then

used for semen analysis. The testes were surgically removed, and suspended in bouins fluid fixation, preparatory

to histological processing.

Table 1. Experimental design

Group No. of Treatment with BLCO Group No. of Treatments with (normal) Animals’

ml/kg b.w (control) Animals saline ml/kg b.w

A 10 0.5 A 5 0.5₁

B 10 1.5 B 5 1.5₁

C 10 2.5 C 5 2.5₁

D 10 3.5 D 5 3.5₁

2.4 Determination Of Sperm Count

The sperm count was determined using the new improved neubauer’s haemocytometer. The homogenate (dilute

semen) was used. Five primary squares were counted; the counting was performed with the help of a hand

counter.

2.5 Statistical Analysis

The results were analyzed for statistical significance by one way and two-way analysis of variance (ANOVA).

All data were expressed as mean ±SEM. P values ˂0.05 were considered significant.

3. Observations And Results

3.1 Sperm Count

The table below (table 2) show data obtained from caudal epididymal sperm count following oral administration

of BLCO. The sperm count was significantly reduced in the treated groups A, B, C, and D; 21.80±0.57,

19.00±0.85, 16.20±0.00, 12.90±0.99 respectively when compared with their corresponding control groups;

26.30±1.27, 25.50±0.71, 26.70±2.69 and 25.60±0.57 respectively at a probability level of P˂0.05. The reduction

in the number of sperm cells was also seen to be dose dependent.

Table 2. Effect of BLCO treatment on sperm count(10 )₁

Group Control Treated

A 26.30±1.27 21.80±0.57*

B 25.50±0.71 19.00±0.85*

C 26.70±2.69 16.20±0.00*

D 25.60±0.57 12.90±0.99*

Values are expressed as Mean±SEM, *=significant at P˂0.05 vs. control

3.2 Histology Of The Testes

The cellular architecture and integrity of the testes were examined in this study, results in the control groups

(shown in micrographs 1, 3, 5 and 7), revealed several layers of spermatogenic cells such as primary

spermatocytes, spermatids, spermatozoa and sertoli cells.

On the other hand, testes of treated rats (micrographs 2, 4, 6 and 8) showed minimal to marked disintegration of

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the basic cellular outline and histology of the seminiferous tubules.

The testes of the treated group A animals showed similar features with the control but fewer spermatids in the

seminiferous tubules. The seminiferous tubules of treated group B rats showed less interstitium and interstitial

cells (of leydig), sertoli cells were found singly. This suggests degenerative and necrotic changes. The testes of

group C animals were similar to those of group B, but the interstitium was scantier and lesser spermatids were

observed. The germinal epithelium of the group D animals were distorted, tubular lamina was scanty with few

spermatids seen.

H AND E STAIN

X100

PLATE 1a Photomicrographs of testes of control rats in Group A

X400 PLATE 1b Photomicrographs of testes of control rats in Group A

H AND E STAIN

Germinal Epithelium

Interstitial Connective

Tissue

Interstitial Cells of Leydig

Blood Vessels

Seminiferous Tubules

Fibroblast

Spermatids

Spermatogonia

Sertoli Cells

Basement Membrane

Primary Spermatocytes

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X100

PLATE 2a Photomicrographs of testes treated with 0.5ml/kg b.w of BLCO rats in Group A1

X400

PLATE 2b Photomicrographs of testes treated with 0.5ml/kg b.w of BLCO rats in Group A1

H AND E STAIN

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X100

PLATE 3a Photomicrographs of testes of control rats in Group B

X400

PLATE 3b Photomicrographs of testes of control rats in Group B

H AND E STAIN

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X100

PLATE 4a Photomicrographs of testes treated with 1.5ml/kg b.w of BLCO rats in Group B1

X400

PLATE 4b Photomicrographs of testes treated with 1.5ml/kg b.w of BLCO rats in Group B1

H AND E STAIN

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X100

PLATE 5a Photomicrographs of testes of control rats in Group C

X400

PLATE 5b Photomicrographs of testes of control rats in Group C

H AND E STAIN

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X100

PLATE 6a Photomicrographs of testes treated with 2.5ml/kg b.w of BLCO rats in Group C1

X400

PLATE 6b Photomicrographs of testes treated with 2.5ml/kg b.w of BLCO rats in Group C1

H AND E STAIN

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X100

PLATE 7a Photomicrographs of testes of control rats in Group D

X400

PLATE 7b Photomicrographs of testes of control rats in Group D

H AND E STAIN

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X100

PLATE 8a Photomicrographs of testes treated with 3.5ml/kg b.w of BLCO rats in Group D1

X400

PLATE 8b Photomicrographs of testes treated with 3.5ml/kg b.w of BLCO rats in Group D1

4. Discussion

This study investigated the effect of ingested BLCO on the sperm count of male wistar rats. Several researches

(Ellenton and Hallet, 1981; Mckee et al., 1994; Eyong, 2000; Didia et al.,2003) carried out in both laboratory

and non-laboratory animals with crude oils of different geologic origins have pointed out the toxic effects of this

important commodity. The observed dose dependent reduction in the number of caudal epididymal sperm cells

of BLCO treated rats is in accordance with earlier reports by Obidike et al., 2007 and Orisakwe et al., 2004. The

observed reduction in the number of sperm cells of the BLCO treated rats suggests depression of spermatogenic

activity which probably indicates a reduction in the number of developing germ cells. These effects can be

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attributed to polyaromatic hydrocarbons (PAH’s) contained in BLCO which has the potential to induce adverse

developmental effects such as termination of pregnancy, malformations, sterility in offsprings, testicular changes

such as lack of sperm, immunosuppression, and tumors (lyons, 1998; Fischer et al., 2007; 2006; 2005).

This study revealed marked changes in the histology of the testes. The degeneration and necrosis of

spermatogenic cells observed in this study corroborates reports in previous studies (Obidike et al., 2007 and

Orisakwe et al., 2004). In addition, the sertoli cells which appeared to lose their connections to the developing

germ cells, probably did not support the maturation and differentiation of these germ cells (Orth et al. 1988).

This probably indicates that BLCO damages the sertoli cells and their number decreases, impairing

spermatogenesis. This leads to a decrease in the number of germ cells with complete differentiation (Steger et al.,

1999). In this study, degeneration and necrosis of the leydig cells following exudation into the interstices was

observed in the testes of the rats that received BLCO.

In conclusion, results from this investigation indicated that BLCO administration can cause deterioration in

semen quality, degenerative and necrotic changes of cells in the seminiferous tubules and interstitium of the

testes as evidenced in the histology of the testes observed. Therefore, haven shown reproductive cyto -toxicity in

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